1. Aim of the Invention
This invention discloses the use of compounds of the azetidinone family in the prevention or treatment of cholesterol gallstone disease of the biliary tree in mammals.
This invention is in the general area of lithiasic disease by cholesterol stones in the biliary tree. In particular, the invention evidences that the use of drugs that specifically block cholesterol intestinal absorption inhibit the onset of cholesterol gallstone disease in the biliary tree. These drugs act by decreasing biliary cholesterol secretion, and at the same time increasing the biliary flow and the hepatic secretion of endogenous compounds (e.g. bile salts, phospholipids) into the bile. In turn, these endogenous compounds contribute to inhibiting cholesterol precipitation and the formation of gallstones in the biliary tree.
2. Background of the Invention
Cholesterol gallstone disease in the biliary tree, specifically in the gallbladder (cholelithiasis), is a highly prevalent illness in the adult population of developed western countries (10 to 15% of the population over 18 years of age). This disease reaches epidemic levels in the Hispanic and Native American populations of most countries in the Andean area, including Chile, Peru, Bolivia and Mexico, among others (Carey and Paigen, 2002; Everhart et al., 2002). In Chile, for example, we have demonstrated that the general occurrence in males is of 17% and in women of 30%, reaching incidences of over 50% in the population over 50 years of age (Miquel et al., 1998a). It has recently been estimated that in the United States, approximately 20 million people had been affected or are currently affected by this disease (Everhart et al., 1999). In countries like Chile, there are more than 2 million habitants affected by cholelithiasis. This epidemiological scenario makes lithiasic disease of the biliary tree the digestive pathology with the highest social cost in countries like the United States, with a annual estimated cost of 8-10 billion dollars, in direct and indirect expenses, which amounts to 1.3-1.5% of the total health care costs (Everhart, 1994). On the other hand, in developing countries such as Chile, cholelithiasisis the first cause of surgical hospitalizations, only surpassed by obstetric hospitalizations, with a high social cost for all the country's health care services (Csendes et al., 1993; Medina, Pascual and Medina, 1983).
Biliary secretion has important physiological functions in our body; among other functions there is the elimination of steroidal molecules by secreting cholesterol and bile salts (catabolic cholesterol products). In fact, the biliary secretion is the main way of eliminating steroidal molecules from the body, having therefore, a central role in cholesterol homeostasis. The pathogenic mechanisms of cholesterol gallstone disease are only partially known. It is accepted as a dogma that the first metabolic defect necessary for the formation of gallstones is the generation of cholesterol rich bile, known as “cholesterol over-saturated bile”, by the liver. The generation of cholesterol over-saturated bile is then a defect in the physiological functions of hepatic lipid metabolism (Carey, 1993; Miquel et al., 1998b). The main metabolic defect in people that develop cholesterol gallstones is the selective increment of cholesterol secretion into the bile, in relation with the molecules that can solubilize cholesterol in the bile (bile salts and phospholipids). Therefore, a thermodynamically unstable bile solution is generated where cholesterol tends to move from a soluble to an insoluble phase, with precipitation of cholesterol crystals and cholesterol gallstone growth, mainly in the gallbladder where local conditions favor stasis and the retention of cholesterol crystals (Apstein and Carey, 1996). It is assumed that there are genetic factors as well as environmental factors involved in the generation of this chronic metabolic disease. However, these factors have yet to be elucidated (Paigen, 2002). We need to emphasize that bile cholesterol is mainly preformed cholesterol derived from plasma lipoproteins and not originated from hepatic neo-synthesis. Nevertheless, it is not clear the participation of cholesterol from the diet (intestinal) and/or cholesterol derived from peripheral tissue to the liver (reverse transport of endogenous cholesterol) in the generation of over-saturated bile and finally of cholesterol gallstones (Paigen, 2002).
Once the gallstones are formed in the gallbladder they are clearly visible by imaging exams such as abdominal sonograms. The gallstones can remain in the gallbladder (or common bile duct) without causing symptoms during the person's whole life (silent gallstones), evolve to simple symptomatic forms (biliary colic type pain), or generate clinical complications with more serious prognosis like acute cholecystitis, vesicular empyema, suppurative cholangitis or acute pancreatitis, among others (Jonston and Kaplan, 1993). At the same time, cholelithiasis is the main risk factor for developing gallbladder cancer, a digestive neoplasia with bad prognosis and elevated prevalence in populations with high incidence of gallstone disease, like those present in Chile, Peru, Mexico, and the Native American and Hispanic populations of North America (Nervi, 2001).
The main risk factors associated with a higher frequency of cholelithiasis in the different populations studied are: female gender, Amerindian genetic background, cholelithiasis in first degree relatives, pregnancy, obesity or overweight in women, abrupt weight loss in the morbidly obese, bariatric surgery in the morbidly obese, diabetes mellitus, puerperium, prolonged fasting, total parenteral nutrition, hypertriglyceridemia (Amigo et al., 1999; Paigen, 2002; Wudel et al., 2002).
The animal models only develop cholelithiasis on the presence of a rich cholesterol diet (approximately 100-1,000 times that of the western human diet). Recently, some genetically manipulated animal models have demonstrated the critical role of the expression of genes relevant in intestinal cholesterol absorption (ACAT-2) and in transport of dietary cholesterol to the liver in the form of chylomicrons (Apolipoprotein E), in the generation of experimental cholelithiasis.
The liver is a central organ in the regulation of cholesterol homeostasis. It is the main organ involved in the synthesis and catabolism of plasma lipoproteins, and the only organ able to eliminate significant quantities of cholesterol (and other steroidal molecules) from the organism, either as free cholesterol (approximately 1 gr/day) (Grundy, 1983) or via its conversion or catabolism into bile salts (Dietschy, Turly and Spady, 1993). Defects in hepatic cholesterol metabolism cause two highly prevalent diseases, atherosclerosis and cholelithiasis. The hepatic cells have complex molecular mechanisms that finally regulate the homeostasis of intracellular cholesterol. Experimental evidence in humans and animals allowed to estimate that the cholesterol destined to biliary secretion is mainly (85-95%) preformed cholesterol generated by the uptake of plasma lipoproteins, and only 5-15% is generated by hepatic biosynthesis (Vlahcevic, 1994). Studies performed in lithiasic patients and controls show contradictory results related to the hepatic cholesterol neo-synthesis activity and bile salts neo-synthesis (review in Apstein and Carey, 1996; Carey, 1993). However, it is accepted that in lithiasic patients, the cholesterol destined to biliary secretion in abnormally high levels is preferentially originated from a preformed pool of lipoproteic origin. The influx of lipoprotein cholesterol towards the liver can come from two sources: a) by reverse transport of cholesterol from peripheral tissues to the liver in HDL and/or LDL (Fielding and Fielding, 1995) and b) cholesterol of exogenous origin (dietary) that is transported from the intestine to the liver fundamentally as remnants of chylomicrons (rQM) and very low density lipoproteins (VLDL) (Wilson and Rudel, 1994). It is important to mention that cholesterol adsorbed at the intestinal level can be of exogenous origin (from the diet) or endogenous (biliary cholesterol). In fact, there is evidence showing that cholesterol present in the bile and secreted into the intestine is qualitatively more relevant (1000 mg/day) that cholesterol present in the western diet (200-400 mg/day). At the same time, cholesterol present in the bile appears to be absorbed more efficiently than dietary cholesterol. Other evidence suggest that in humans, dietary or intestinal cholesterol could be very relevant in the generation of lithogenic bile in people genetically predisposed to develop this disease. Most of the cholesterol deposited in gallstones seems to have a dietary origin (Holzbach, 1984). In addition, other evidence support the idea that cholesterol of intestinal origin is preferentially destined to biliary secretion facilitating the formation of lithogenic bile (Cooper, 1991). For example, a cholesterol enriched diet in gallstone carriers significantly increases the biliary cholesterol content in the subsequent weeks (Kern, 1994). There is only one repot that has compared the effect of dietary cholesterol in gallstone women and controls, demonstrating that only gallstone patients increase the biliary cholesterol secretion when exposed to an exogenous overcharge of dietary cholesterol (Kern, 1994). This data suggests that dietary cholesterol that can be destined to biliary secretion is probably regulated by yet unidentified genes, and their regulatory mechanisms appear to be altered in patients that develop gallstone disease.
In contrast to the almost complete intestinal absorption of the majority of other nutrients, cholesterol absorption is limited to an average of 40-60% of the ingested cholesterol, with great variability among different species (Jolley, Dietschy and Turley, 1999) and amid people (Gylling and Miettinen, 2002b). The variability in cholesterol absorption capacity among individuals can be as high as 4-6 times (in a range of 30-80%) for a similar amount of dietary cholesterol. This finding contrast with the intra-individual variations observed at different times under the same dose of dietary cholesterol (<6%) (Bosner et al., 1993). Similar variations in dietary cholesterol absorption ability can be observed in animal models, especially among different mice strains (Jolley et al., 1999). In the last few years, this variability in the capacity to adsorb dietary cholesterol has attained great relevance, not only from the viewpoint of studying the physiology of cholesterol metabolism, but also from the perspective of designing more effective therapeutic alternatives for the treatment of dyslipidemias and atherosclerosis (Gylling and Miettinen, 2002a). During the last years, it has been possible to unravel, at least partially, the molecular mechanisms that control and regulate cholesterol intestinal absorption (Klett and Patel, 2004).
The development of compounds that interfere with cholesterol absorption has gain great relevance during the last years with the aim of developing strategies for the treatment of hypercholesterolemias and atherosclerosis. The compounds that have been evaluated can be classified in three categories depending on their site of action: A) Those that interfere with intestinal absorption at the intra-luminal level, such as resins like cholestyramine, and natural and synthetic saponines, B) drugs that interfere with the entry of cholesterol into the enterocyte, such as type 2-azetidinones (Ezetimibe) and C) drugs that interfere with the esterification of cholesterol incorporated into the enterocyte, specifically inhibiting the enzyme ACAT-2. Only the compounds mentioned in point A and the 2-azetidinones are currently used in clinical practice for the treatment of dyslipidemias and prevention of cardiovascular diseases.
EZETIMIBE (SCH 58235, ((−)-1-(4-fluorophenyl)-(3R)-[3-(4-fluorophenyl)-(.S)-hydroxypropyl]-(4S)-(4-hydroxyphenyl)-2-azetidinone)) is the first drug in a group of hypocholesterolemic medicines, the specific inhibitors of cholesterol absorption (Klett and Patel, 2004), derived from the structural class of the 2-azetidinones. It was approved by the US Food and Drug Administration in October 2002, under the registered brand Ezetrol®-MSD-Schering-Plough; and Zetia®, Merck/Schering Plough, North Wales, Pa. Ezetimibe (SCH 58235) corresponds to the second generation of specific inhibitors of cholesterol intestinal absorption, and was generated by structural modification and metabolites of SCH 48461 and SCH 53695 (SCH 53695 is the C-4 phenol of SCH 48461). Ezetimibe is a metabolite derived from the active forms of SCH 48461, with an inhibitory activity in cholesterol absorption 400 times higher than its predecessor analog (SCH 48461). This increase in the inhibitory activity of cholesterol absorption has been attributed to the incorporation of phenolic rings into SCH 48461 (Jeu and Cheng, 2003). Ezetimibe has demonstrated to effectively inhibit intestinal cholesterol absorption in mammalian models (murine models) in dosages that fluctuate between 0.1-6 mg/kg body weight per day. Ezetimibe is indicated to be used as monotherapy or as combined therapy with inhibitors of the HMG-CoA-reductase (statins) in the treatment of primary hypercholesterolemias for the reduction of total cholesterol, LDL cholesterol and ApoB. It is also indicated as combined therapy in patients with homozygous familial hypercholesterolemia, and as monotherapy in patients with homozygous familial sitosterolemia (Cheng and Leiter, 2003; Davidson, 2003; Iglesias and Diez, 2003; Lipka, 2003). The combined therapy with 2-azetidinones associated to inhibitors of the HMG-CoA-reductase (statins) such as pravastatin, simvastatin, rosuvastatin, lovastatin, fluvastatin, atorvastatin and cerivastatin, is based in the selective inhibition of cholesterol intestinal absorption. Because of this, a reduction in the hepatic cholesterol content triggers a compensatory associated effect in humans, increasing the neo-synthesis of cholesterol at the hepatic level. Therefore, when the 2-azetidinones are associated with a selective inhibitor (statins) of the critical enzyme (HMG-CoA-reductase) in the cholesterol neo-synthesis pathway, the reduction in the plasma cholesterol is enhanced. The combined therapy with 2-azetidinones plus inhibitors of the HMG-CoA-reductase in the pathology concerned in this invention (cholelithiasis) has not been evaluated and there are no communications about it in the scientific and medical literature.
The specific mechanism of Ezetimibe action as a selective inhibitor of cholesterol absorption is still not known in detail. It is hypothesized that it acts by inhibiting the entry of cholesterol from the lumen into the enterocyte, acting at the apical plasma membrane level and interacting with some proteins involved in cholesterol trafficking (Klett and Patel, 2004). Ezetimibe is incorporated into the enterocyte, is glucuronized into its active metabolite at this level, and enters the entero-hepatic circuit of lipids (Jeu and Cheng, 2003). Even though Ezetimibe is up taken with high affinity by the liver, and is actively secreted toward the biliary tree reaching elevated concentrations in the bile, it is not known if this drug has other additional functions in the transport of steroidal molecules (e.g. cholesterol) at the hepatic and biliary tree levels.
It is universally accepted today that the only effective therapy for the treatment of gallstone disease is the surgical resection of the gallbladder with its gallstones (cholecystectomy) (Gui et al., 1998). However, some pharmacological alternatives for treatment (non-surgical) have been developed. These treatments demonstrated effectiveness in the dissolution of gallstones in specific subgroups of patients with cholelithiasis (Hillebrant et al., 2002; Stiehl et al., 1984) and in some specific groups of patients at high risk for developing gallstone disease (Mason and Renquist, 2002; Wudel et al., 2002).
The medical alternative is the dissolution of cholesterol gallstones by bile salts, chenodeoxycholic and ursodeoxycholic acid (Sugata, 1993). The most utilized internationally has been ursodeoxycholic acid because it shows lower rates of adverse effects and similar efficacy. These drugs act by increasing the bile salts contents in the bile and decreasing hepatic cholesterol secretion, thus generating bile that is not supersaturated with cholesterol and allowing the solubilization of cholesterol gallstones. It was demonstrated to be an affective therapy in patients with small cholesterol gallstones. However, it requires long therapy periods (6-12 months) and it is associated with high recurrence levels (50% in 5 years) when discontinued (Bilhartz, 1998; Stiehl et al, 1984). The association of ursodeoxycholic acid with statins (inhibitors of cholesterol neo-synthesis) has also been explored to this same end, but the efficacy of the therapy has not been demonstrated (Hillebrant et al., 2002; Miettinen et al., 1998).
The possible utility of other hypolipaemiant drugs in the treatment of cholesterol gallstone disease has been evaluated without any clinical demonstrated effect. The use of fibrates, niacin or resins that decrease cholesterol and bile salts intestinal absorption (cholestyramine, probocol), has demonstrated no therapeutic effectiveness in cholesterol gallstone disease patients. Moreover, the use of hypolipaemiant therapies (that decrease plasma cholesterol) with fibrates (specifically clofibrates) in humans shows a clear increase in the risk of developing cholelithiasis (Amigo et al., 1999; Apstein and Carey, 1996). On the other hand, the use of HMG-CoA-reductase inhibitors (statins) has shown contradictory results in pre-clinical and clinical models. In some experimental models and in some but not all human studies, this therapy reduces the biliary cholesterol content and the lithogenicity index; however it has not been demonstrated to be useful in the dissolution of cholesterol gallstones in prospective clinical studies (Chapman et al., 1998; Hillebrant et el., 2002; Porsch-Ozcurumez et al., 2001).
The main problem is that nowadays there is no form for efficient primary prevention of gallstone disease in the general population or in specific high-risk groups (women, third trimester of pregnancy, puerperium, overweight or obese women, programmed weight loss in the morbidly obese, gastroplasty and intestinal by-pass in the morbidly obese, prolonged fasting, prolonged enteral nutritional therapy); nor there are effective medical therapies (e.g. dissolution of already formed gallstones).
It is known that the 2-azetidinones and their respective pharmacological families are used in the treatment of elevated plasma cholesterol and are protected by diverse patents. For example, the documents U.S. Pat. No. 5,631,365, WO 2004 010948, WO 2004 081002, WO 2004 000803, WO 2004 000804, WO 2004 000805, divulge the use of Ezetimibe for the treatment of cholesterol disorders such as hypercholesterolemia, atherosclerosis, or cholesterol induced tumors.
Even though cholelithiasis is a disease of cholesterol metabolism, there is not a clear correlation between total cholesterol plasma levels, LDL cholesterol plasma levels or atherosclerosis with the presence of cholesterol gallstones or with a higher risk of developing cholelithiasis. Consequently, the different therapies developed for the treatment of hypercholesterolemia or dyslipidemias, main risk factors of atherosclerotic cardiovascular illness, have not been demonstrated to be effective in the treatment and/or prevention of cholesterol gallstone disease. Only hypertriglyceridemia and low plasma levels of HDL cholesterol have been associated in some studies with a higher risk of gallstone disease, without demonstrating a causal relationship between these serological variables and the development of cholelithiasis (Paigen, 2002).
Because of the arguments presented above is that the present invention will solve a problem with the technique, since it is not an obvious inference that the current techniques developed for the treatment of plasma dyslipidemias and/or atherosclerosis are effective in the treatment of gallstone disease.
A 2-azetidinone compound in combination with other drugs and its use in the treatment of cholesterol gallstone disease or cholelithiasis is mentioned in the documents WO2004001002 and WO20030153541. These documents specifically proclaim a pharmaceutical composite that includes a moderator of a LXR receptor and an agent of lipidic regulation such as Ezetimibe. The orphan nuclear receptor LXR acts a transcriptional regulator of genes involved in cholesterol metabolism and trafficking. When it binds endogenous ligands (oxysterols) or synthetic ligands it inhibits cholesterol synthesis (specially in the liver) and at the same time stimulates cholesterol catabolism into bile salts and cholesterol exit into the bile in rodents (Yu et al., 2003). It was demonstrate in experimental animal models (rodents) that the activation of this nuclear receptor lowers cholesterol plasma levels, particularly under cholesterol rich experimental diets that increase cholesterol content in mice bile by activating specific genes known as ABCG5 and ABCG8. This findings have not been demonstrated in humans yet since there are no drugs approved for human use. The available evidence from experimental animals suggest that the use of LXR activating drugs could also increase the risk of cholesterol gallstone formation in the biliary tree since the stimulation of LXR increases cholesterol content in the bile (Yu et al., 2003). There is no evidence in the literature showing that the use of LXR synthetic ligands (pharmacological), alone or in combination with Ezetimibe, inhibit the formation of cholesterol stones of the biliary tree.
The document WO2005 00217 divulges a compound that combines an anti-obesity agent and an anti-dyslipidemiant agent, among which there is an inhibitor of cholesterol absorption like Ezetimibe. This composite is indicated for its use in dyslipidemia-related disorders. The document WO2004078716 describes piperazine derivatives and their use as agonists of the melanochortin receptor (MC-R), indicated for the treatment of obesity. Additionally, document WO2004030637 discloses the use of modulators of the metabotropic glutamate receptor 5 (mGluR5) in the treatment of obesity related disorders. In the description they mention that “anti-obesity agents apt to be used in combination with a modulator of the mGluR5 include, among other cholesterol lowering agents, cholesterol absorption inhibitors such as 2-azetidinones like Ezetimibe”. Document WO2004031175 describes compounds that act as agonists of NPY receptors, specifically NPY Y5 receptors. This compounds are useful for the treatment of diverse ailments related to NPY, being among them gallbladder disease. Obesity and associated medical problems such as gallstone disease can also be treated. Ezetimibe is mentioned as an example of other active ingredients that can be administered in combination with the compound related to the invention.
Overweight (body mass index, BMI, >25 kg/mt2) and obesity (BMI>28) are established risk factors for developing cholelithiasis mainly in women, being this evidence more clear than in men. Overweight and obesity increase the risk of cholelithiasis 2-3 times in women but not in men. On the other hand, abrupt weight loss (>1.5 kg/week) in the morbidly obese (BMI>35), either by hypocaloric dietary treatment (approximately 1,000 kcal/day) or though bariatric surgery, increases the risk of developing cholelithiasis, and it has been reported that 20-40% of these patients can develop cholelithiasis in the subsequent weeks or months. It is not clear why only some overweight or obese subjects develop cholelithiasis (20-30% according to some studies), and other environmental and/or genetic factors that may determine this susceptibility have been suggested (Paigen, 2002 and references therein).
Nevertheless, most of the people that develop cholelithiasis are not overweight or obese and therefore this association is far from being universal. It is probable that the treatment of obesity per se with the current techniques could even increase the risk of developing cholelithiasis in these subjects. On the other hand the prevention of this metabolic condition could lower, at least in part, the frequency of this disease in high risk populations (genetically susceptible).
Because of the arguments presented above is that the present invention will solve a problem with the technique, since it is not obvious to infer that the current techniques developed for the treatment of morbid obesity and/or overweight are effective in the treatment of gallstones disease.